The Order Kinetoplastida contains several pathogens of medical importance. These organisms employ an unusual mechanism of gene expression that requires the trans-splicing of every nuclear mRNA with the 39-nt spliced leader (SL) RNA. This RNA and mode of splicing is not found in humans and represents a unique target for chemotherapy. The SL RNA receives a m7G cap co-transcriptionally, followed by the addition of seven methylations on the sugar and/or base moieties of the first four nucleotides, constituting the unique 'cap 4'. Undermethylated substrate SL RNA is trafficked intracellularly prior to trans-splicing in Trypanosoma brucei and Leishmania tarentolae, introducing potential competitors into the cytosol for translation initiation factors. The basis of this application is a new addition to our hypothesis on the maturation of SL RNA involving a 'mask'protein that prevents eIF4F translation-initiation complex formation on the unspliced substrate. A unifying explanation for the undermethylation and loss of polysome formation for SL RNA mutants in L. tarentolae, which generally exhibit the cap 1 phenotype, is that they are recognized by the translational masking factor, but cannot be unmasked. The inability of these SL mutants to remove the translational mask is that 1) their 5'ends are not accessable for subsequent cap 2-4 methylations and 2) once they are trans-spliced, they are still translationally masked and either not exported from the nucleus or not loaded onto polysomes in the cytosol. The level of discrimination that exists among known cap-binding proteins has led us to search for this SL mask in the families of eIF4F factors. The hypothesis underlying these experiments is that immature SL RNA must be masked from the translation machinery so that the incomplete 5'-cap structure does not interfere with protein synthesis. The presence of six potential homologs of the cytosolic cap-binding protein eIF4E and six of associated translation initiation factor eIF4G in T. brucei provided candidates for examination;preliminary data indicates that TbeIF4E-3 fits the SL mask requirements. The specific aims of this application are: 1) To validate the cap-binding properties of TbeIF4E-3, a homolog of the Leishmania major cap 0-binding protein, and/or other candidates;and 2) To determine the TbeIF4E-3 RNA substrate(s) and any associated proteins. These studies will lead to a thorough understanding of kinetoplastid SL biogenesis and the interplay between RNA processing and translation. PUBLIC HEALTH RELEVANCE: The Order Kinetoplastida contains several pathogens of medical importance. These organisms employ an unusual mechanism of gene expression that requires the transsplicing of every nuclear mRNA with the 39-nt spliced leader (SL) RNA. This RNA and mode of splicing is not found in humans and represents a unique target for chemotherapy.